Browsing by Author "Roepcke, Clarisse Brüning Schmitt"

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Open Access
Development of acetylcholinesterase biosensors for neurotoxins detection in foods and the environment
(2011) Roepcke, Clarisse Brüning Schmitt; Hauer, Bernhard (Prof. Dr.)
Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the nervous system. It is inhibited by several substances, like organophosphate and carbamate insecticides, glycoalkaloids, nerve gas and anatoxin-a(s). There is a growing need to develop new technologies to reduce time spent with sample preparation, discriminate between positive and negative samples, and to reduce analysis costs. Over the last decades, AChE biosensors have emerged as an ultra sensitive and rapid technique for toxicity analysis in environmental monitoring, food and quality control. Acetylcholinesterase is only slightly inhibited by organophosphorothionate insecticides, the most applied organophosphate insecticides worldwide. This fact makes their detection analytically very difficult. A new enzymatic method for the activation and detection of phosphorothionates was developed with the capability to be used directly in food samples without the need of laborious solvent extraction steps. Chloroperoxidase (CPO) from Caldariomyces fumago was combined with tert-butyl hydroperoxide and two halides. Chlorpyrifos and triazophos were completely oxidized. Fenitrothion, methidathion and parathion methyl showed conversion rates between 54 – 61%. Furthermore, the oxidized solution was tested with an AChE biosensor assay. Chlorpyrifos spiked in organic orange juice was oxidized, and its oxon product was detected in concentrations down to 5 µg/L (final concentration food sample: 25 µg/L). The complete duration of the method took about 2 h. An acetylcholinesterase B multisensor from Nippostrongylus brasiliensis (Nb) was developed to detect the most frequently used insecticides in Brazil. The objective was to establish a fast screening method, separating the negative samples from the positive ones. The four mutants, which together presented the widest sensitivity spectrum, were: F345A, M301A, W346V and W346A. The combination of these four mutants in a multienzyme biosensor array enabled the detection of 11 out of the 12 most important insecticides at concentrations below 20 µg/L. The biosensor test was compared with traditional analysis methods, and validated with food samples previously analyzed. The storage stability revealed that the biosensor remained stable for 40 weeks; however the sensitivity decreased with time. Glycoalkaloids are secondary metabolites present in potatoes, which can be toxic to humans in high concentrations. Alfa-Solanine and alfa-chaconine are the main examples of this group, and these substances show an anti-acetylcholinesterase activity. An Nb acetylcholinesterase B biosensor was designed to detect glycoalkaloids in buffer solutions and in foods. The two Nb AChE mutants that showed the highest sensitivity towards alfa-solanine and alfa-chaconine (lowest I50 value) were W303L and F345A. The Dixon and Cornish-Bowden plots demonstrated that the inhibition of these substances over Nb AChE was reversible and competitive. The achieved detection limits of alfa-chaconine and alfa-solanine were 0.1 and 0.5 µM, respectively. The designed biosensor was able to detect mixtures of alfa-solanine and alfa-chaconine in potatoes samples spiked with these glycoalkaloids in total concentrations higher than 300 µM.